Regulation of Abiotic Stress Responses in Plants by the Ubiquitin Pathway

该项目旨在了解植物如何应对压力环境以生存。虽然已知植物在暴露于诸如寒冷、干旱和高盐度等条件时会合成一种新的化学物质，但不了解的是植物如何“清理房屋”，即如何消除压力信号，以便一旦压力消失，植物可以开始正常生长。遗传学和生物化学方法将被用来确定这些变化是如何发生的。这项研究将通过研究项目提供多个UC-Davis本科生的培训，并支持研究生和博士后学者。这些人将学习科学方法以及如何向同事和更大的社区提供科学数据。他们将学习分析技能，提高他们的口语和写作能力。研究生将有机会与K-12学校在邻近社区与大量的西班牙裔人口互动。研究人员将通过加州大学戴维斯分校的种子研究所与加州种子/农业生物技术行业的代表进行互动。研究人员已经与当地的一位萨克拉门托高中教师建立了联系，并将继续这种合作。高中生将学习实验设计和数据分析，并参与科学发现。植物通过上调基因表达来应对环境压力。应激反应所需的一个转录因子家族被称为ABRE结合蛋白（ABF），并在蛋白质水平上受到调节。在压力下，它们被激活以促进转录，同时它们的降解停止，蛋白质水平增加。压力如何导致ABF的这两种变化以及它们与生物功能的关系尚不清楚。将通过体外和体内降解试验确定负责调节正常生长条件下组成型ABF蛋白水解和应激诱导蛋白稳定的ABF氨基酸。编码ABF蛋白的DNA序列将被改变，因此它们翻译具有缺失和/或氨基酸取代的ABF蛋白。ABF片段也将与标记酶的编码区融合，并在快速和定量测定中确定融合的稳定性。改变的蛋白质将从细菌中表达和纯化，并在植物裂解物中测量稳定性。将序列引入植物中，产生表达这些蛋白质的多个稳定系，并测量体内蛋白质稳定性。通过测量预测或可能的磷酸化位点被丙氨酸（不能修饰）或天冬氨酸（模拟修饰）取代的蛋白质的稳定性，评估磷酸化状态的作用。将评估表达具有降解速率差异的ABF蛋白的转基因植物在正常和胁迫条件下的生长表型，以确定降解的调节是否提供更大的胁迫抗性而不损害正常生长。

This project aims to understand how plants respond to a stressful environment in order to survive. While it is known that plants synthesize a novel chemical when exposed to conditions such as cold, drought and high salinity, what is not understood is how the plant "cleans house", that is, how the stress signal is removed so that the plant can begin normal growth once the stress is gone. Both genetic and biochemical approaches will be taken to determine exactly how these changes happen. This research will provide training of multiple UC-Davis undergraduates through research projects and support a graduate student and a post-doctoral scholar. These individuals will learn scientific methodology and how to present scientific data to colleagues and the greater community. They will learn analytical skills and improve their speaking and writing abilities. The graduate student will have the opportunity to interact with K-12 schools in neighboring communities with large Hispanic populations. The researcher will interact with representatives of the California seed/agricultural biotechnology industries through the Seed Institute at UC-Davis. The researcher has established a connection with a local Sacramento high school teacher and will continue this collaboration. The high school students will learn experimental design and data analysis, and participate in scientific discovery. Plants respond to environmentally stressful conditions by upregulating expression of genes that help them cope. One family of transcription factors required for stress responses are called ABRE BINDING PROTEINS (ABFs), and are regulated at the protein level. Under stress they are activated to promote transcription and at the same time their degradation is stopped and protein levels increase. How stress causes these two changes in ABFs and their relationship to biological function are not understood. ABF amino acids responsible for regulating constitutive ABF proteolysis under normal growth conditions and for stress-induced protein stabilization will be determined through in vitro and in vivo degradation assays. DNA sequences encoding ABF proteins will be changed so they translate ABF proteins with deletions and/or amino acid substitutions. ABF segments will also be fused with coding regions of marker enzymes and stability of the fusion determined in rapid and quantitative assays. Altered proteins will be expressed and purified from bacteria and stability measured in plant lysates. Sequences will be introduced into plants, multiple stable lines expressing these proteins produced and the in vivo protein stability measured. The role of phosphorylation status will be assessed by measuring the stability of proteins with substituted predicted or possible phosphorylation sites with either alanine, which cannot be modified, or with aspartate, which mimics modification. Transgenic plants expressing ABF proteins with differences in degradation rates will be assessed for their growth phenotypes under normal and stress conditions to determine if modulation of degradation provides greater stress resistance without compromising normal growth.